2,7-naphthyridinone-based MET kinase inhibitors: A promising novel scaffold for antitumor drug development

Article abstract of DOI:10.1016/j.ejmech.2019.06.033

As part of our effort to develop new molecular targeted antitumor drug, a novel 2,7-naphthyridone-based MET kinase inhibitor, 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)- 3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one (13f), was identif

Full text of DOI:10.1016/j.ejmech.2019.06.033

Accepted Manuscript
2,7-naphthyridinone-based MET kinase inhibitors: A promising novel scaffold for
antitumor drug development
Lin-Sheng Zhuo, Hong-Chuang Xu, Ming-Shu Wang, Xing-E. Zhao, Zhi-Hui Ming,
Xiao-Lei Zhu, Wei Huang, Guang-Fu Yang
PII:
S0223-5234(19)30561-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.033
EJMECH 11436
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2019
Revised Date: 7 June 2019
Accepted Date: 12 June 2019
Please cite this article as: L.-S. Zhuo, H.-C. Xu, M.-S. Wang, X.-E. Zhao, Z.-H. Ming, X.-L. Zhu, W.
Huang, G.-F. Yang, 2,7-naphthyridinone-based MET kinase inhibitors: A promising novel scaffold
for antitumor drug development, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract
2,7-Naphthyridinone-Based MET Kinase Inhibitors: A
Promising Novel Scaffold for Antitumor Drug Development
Lin-Sheng Zhuo1,†, Hong-Chuang Xu1,†, Ming-Shu Wang1,†, Xing-E Zhao2, Zhi-Hui
Ming2, Xiao-Lei Zhu1, Wei Huang1,*, and Guang-Fu Yang1,*
cyclization
O
bioisosterism
N
N
O
N
Block D
F
H
H
N
N
Block B
scaffold
hopping
F
NH
O
scaffold
hopping
(b)
NH
O
F
F
F
O
O
Block C
(a)
O
O
O
Block C
13f
O
O
Cl
(2,7-naphthyridinone)
Cl
H2N
BMS777607
Cabozantinib
Good in vitro potenvy:
IC50 = 9.9 nM (c-Met), 69.0 nM (MKN -45);
Orally exposed: F = 54% (rat);
N
H2N
N
N
Block A
Excellent in vivo efficacy: MED = 12.5 mpk.
TGI = 114% & 6/6 PR (50 mpk, U-87 MG)

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2,7-Naphthyridinone-Based MET Kinase Inhibitors: A Promising
Novel Scaffold for Antitumor Drug Development
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4
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Lin-Sheng Zhuo1,†, Hong-Chuang Xu1,†, Ming-Shu Wang1,†, Xing-E Zhao2, Zhi-Hui Ming2,
Xiao-Lei Zhu1, Wei Huang1*, and Guang-Fu Yang1,*
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1Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensor Technology and Health, College of Chemistry, Central China
Normal University, Wuhan 430079, P.R. China
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9
10
2Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, Jiangsu Simcere
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12
Pharmaceutical Co. Ltd, Nanjing 210042, P. R. China
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* Corresponding authors. E-mail address: weihuangwuhan@126..com (W. Huang),
gfyang@mail.ccnu.edu.cn (G. Yang).
†
L.Z.,H.X.and M.W. contributed equally to this work.
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ABSTRACT: As part of our effort to develop new molecular targeted antitumor drug, a novel
2,7-naphthyridone-based MET kinase inhibitor, 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-
3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one (13f), was identified.
Knowledge of the binding mode of BMS-777607 in MET led to the design of new inhibitors
that utilize novel 2,7-naphthyridone scaffold to conformationally restrain the key
pharmacophoric groups (block C). Detailed SAR studies resulted in the discovery of a new
MET inhibitor 13f, displaying favorable in vitro potency and oral bioavailability. More
importantly, 13f exhibited excellent in vivo efficacy (tumor growth inhibition/TGI of 114 %
and 95 % in 50 mg/kg, respectively) both in the U-87 MG and HT-29 xenograft models. The
favorable drug-likeness of 13f indicated that 2,7-naphthyridinone may be used a promising
novel scaffold for antitumor drug development. The preclinical studies of 13f are under way.
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KEYWORDS: 2,7-Naphthyridone, MET kinase inhibitor, Antitumor, In vivo efficacy
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1. Introduction
MET is a heterodimeric transmembrane receptor tyrosine kinase (RTK) with a unique
structure that is distinct from other RTK families.1,2 Upon ligand (hepatocyte growth factor,
HGF) binding, MET induces several complex signaling pathways resulting in cell
proliferation, motility, migration, and survival.3,4 MET addiction, expedience and inherence
have been reported to be associated with cancer onset, progression and therapeutic
response.5,6 Therefore, MET kinase has been considered as an attractive target for molecular
targeted therapy in cancers.
H
H
N
N
Block B
Block D
F
N
N
O
O
O
N
N
Block C
N
O
O
N
N
N
N
N
1
2
Block A
(savolitinib)
(cabozantinib)
9
10
Figure 1. Examples of selective and multi-targeted MET inhibitors
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Several MET-targeting agents, including HGF and MET antibodies, as well as small
molecule kinase inhibitors, are currently in early or advanced stages of clinical testing.6-9
Although many structurally diverse small molecule MET kinase inhibitors have been
reported, ATP-competitive MET inhibitors can be categorized into two groups based on their
chemotypes and binding modes in the MET kinase pocket. Class I inhibitors adopt a U-shape
conformation (DFG-in conformation) and interact with the ATP-binding site at the pocket
entrance, whereas class II inhibitors bind to MET with a linear conformation (DFG-out
conformation) extending from the ATP-binding site to Ile1145 near the C-c-spiral block.
Due to their different binding modes, class I and II inhibitors display significantly different
characteristics. Compared with class II inhibitors, class I inhibitors have high specificity for
MET. For example, Savolitinib (1), a selective class I MET inhibitor is in late-stage clinical
trial (Figure 1).10 Cabozantinib (2), a class II inhibitor, the first small molecule multitargeted
kinase inhibitor of MET, VEGFR-2 and RET was approved by FDA for the treatment of
patients with progressive metastatic medullary thyroid cancer (2012), advanced kidney
cancer (2016) and liver cancer who have previously been treated with the medicine sorafenib
(2018).11
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Figure 2. Reported derivatives of Cabozantinib designed by the cyclization strategy of block C
3
4
As shown in Figure 1, The molecular structure of Cabozantinib (2) can be divided into
four blocks known as A, B, C and D. Among them, the cyclization of block C is the most
successful strategy to develop new MET inhibitors (Figure 2). For example, we reported a
new class of pyridine-based class II MET inhibitors with high potency and selectivity by the
C-1 cyclization strategy.18 It's obvious that the key C-3 cyclization strategy resulted in the
discovery of many new MET inhibitors bearing pyridone, pyridine, pyrimidinone, pyrimidine,
pyridazine, pyrazole, imidazole, triazole cycle and corresponded fused heterocycle, including
several clinical candidates, such as BMS-777607 (IV), merestinib (IV), BMS-794833 (VIII),
G-773 (IX), RXDX-106 (X), AMG-458 (XVIII) and ningetinib (XVIII).
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6
7
8
9
10
11
12
13
Figure 3. Design of 2,7-naphthyridinone derivatives as new MET inhibitors based on BMS-777606
14
15
16
17
18
19
20
Compound 3 (BMS-777607), which exhibits desirable drug-likeness and modest
selectivity to VEGFR-2, was selected as a starting point for our research.13 Analysis of the
crystal structure of 3 and the MET kinase complex (PDB code, 3F82) revealed that the
carbonyl group in pyridone (block C) forms an intramolecular hydrogen-bond (H-bond) with
the amide N-H group and a second H-bond with the backbone N-H of Asp1222. The
pyridone-based block C assumes a pseudocyclic conformation with maximum deviation from
planarity of 0.06 Å in the nitrogen atom of amide group. Herein, we proposed a
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scaffold-hopping strategy that utilizes 2,7-naphthyridone to conformationally restrain key
pharmacophoric groups within block C of the molecule (Figure 3). Furthermore, the structure
activity relationship (SAR) studies of the block A and block D were carried out to optimize
drug-likeness.
5
6
2. Chemistry
By the retro-synthetic analysis, the assembly of chlorinated 2,7-naphthyridone 4 (block
C-D) with aromatic amine 5 (block A-B) was designed to generate target molecule (Scheme
1). As diverse aromatic amine 5 could be easily obtained by the reported methods,19-21 the key
point in the synthesis of target compounds was the construction of
8-chloro-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one 4. There were two potential
approaches to synthesize 4 according to the cyclization order of the 2,7-naphthyridine ring
(Scheme 1). Method A: pyridinone 6 was first chlorinated to generate tri-substituted pyridine
7. Naphthyridine 9 was prepared by the condensation of 7 with DMF-DMA, followed by the
cyclization of 8 under H2SO4. But we failed to introduce the 4-fluorophenyl group into 9 by a
common Buchwald cross-coupling reaction. Under the Pd-catalyzed conditions, the
dehalogenation was the main reaction, due to the high reactivity of the 8-chlorine atom in
naphthyridine, thus making it difficult to obtain 4.
7
8
9
10
11
12
13
14
15
16
17
Method A
Me
Me
NC
Me
NC
N
i)
Me
ii)
iii)
N
NH
N
NH
N
NC
Cl
O
Cl
7
O
9
Cl
6
8
Method B
iv)
iv)
Me
Me
NC
N
Me
ii)
iii)
N
N
i)
HN
N
N
N
NC
O
4
Cl
O
O
12
F
O
O
F
F
F
10
11
NH2
F
v)
C
Method A
Method B
O
R2
R1
N
N
X
D
N
2'
N
5
B
7'
O
N
NH
for
F
F
N
N
Cl
O
F
O
N
13
R2
R1
A
X
R1 = CN
R1 = NH2
vi),vii)
13f-h
18
19
20
21
Scheme 1. Reaction conditions and reagents: i) POCl3, 110°C; ii) DMF-DMA, DMF, 90°C; iii) H2SO4,
110°C; iv) CuI, DMEDA, K3PO4, dioxane, 100°C; v) Pd2(dba)3, dppp, t-BuONa, dioxane, 100°C; vi)
TEMPOH, DCM, 40°C; vii) PhI(OAc)2, ACN, H2O, 0°C.
22
After re-analysed the molecular structure of 4, we found that the nitrogen atoms at 2- and
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7-positions were symmetric in the naphthyridine scaffold. Therefore, we modified the
synthetic route and developed Method B to construct a chlorinated pyridine unit of
2,7-naphthyridine in the last step. The Ullmann-type coupling of pyridone 6 with
1-fluoro-4-iodobenzene produced the 1-phenyl pyridine-2-one unit of 2,7-naphthyridine (10).
The condensation of 10 with DMF-DMA followed by H2SO4-mediated cyclization and
subsequent POCl3-based chlorination successfully yielded the key block C-D intermediate 4.
With intermediate 4 in hand, compounds 13a-h were successfully synthesized through
direct palladium-catalyzed coupling reactions of 4 and the corresponding aromatic amine 5. It
should be pointed out that, to synthesize 13a-c bearing a C(2) amino group in the pyridine
ring in block A, a cyano group was firstly introduced into the 2- position to improve the
coupling reaction. The cyano group was then converted to an amide group with TEMPOH.
However, subsequent conventional Hofmann degradation of amide derivatives using typical
reagent Br2-NaOH22 resulted in a complicated mixture. After optimization, iodo-acetate
benzene was eventually identified as an effective reagent with the advantages of good yields,
short times, mild conditions and ready isolation of the products.23
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Scheme 2. Reaction conditions and reagents:: i) 2,4-pentanedione, piperidine, EtOH, 90°C; ii)
DMF-DMA, DMF, 90°C; iii) H2SO4, 110°C; iv) POCl3, 110°C; v) 9, Pd2(dba)3, dppp, t-BuONa, dioxane,
100°C; vi) TEMPOH, DCM, 40°C; vii) PhI(OAc)2, ACN, H2O, 0°C.
18
19
20
21
22
23
24
25
As shown in Scheme 2, the one-step condensation of 2-cyano-N-phenylacetamide 14 with
2,4-pentanedione produced the key pyridine-2-one intermediate 15 in which the N(1) phenyl
group in block D was successfully introduced. Similar to the synthesis of 13, compound 19
was ultimately produced by a condensation-cyclization-chlorination-coupling reaction of 15.
The abolishment of the copper-catalyzed Ullmann-type coupling reaction in the introduction
of block D greatly improved the synthetic convenience of 19.
26
27
In addition, the crystal structure of 13f was determined by X-ray diffraction analysis.24 In
the crystal structure, the block C of 13f shows good co-planarity, with a max deviation from
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planarity of 0.05 Å at N(1) atom. The plane of the 2,7-naphthyridinone and phenyl ring of
block B were nearly parallel, with a dihedral angle of 3.5o.
3
4
3. Results and discussion
Table 1. Activity of 13a-h and 19a-h against MET and VEGFR-2a
5
6
IC50, nM
MET
enzyme-baseda
132.5
664.2
151.9
418.8
192.0
9.9
Compd.
Block A
Block D
VEGFR-2
enzyme-baseda
cell-basedb
c
13a
13b
13c
13d
13e
13f
A-1
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-6
A-7
A-6
A-6
A-6
A-6
A-7
A-7
3
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
4-F-Ph-
Ph-
-
-
-
-
-
-
-
-
-
-
69.0
279.9
63.2
-
13g
13h
19a
19b
19c
19d
19e
19f
10.7
116.7
103.0
1.5
-
89.8
261.1
102.6
23.1
147.9
44.9
-
2.4
117.8
18.0
-
2,4-di-F-Ph-
4-Cl-Ph-
4-CF3O-Ph-
Ph-
30.5
-
33.2
-
>1000
5.5
-
19g
19h
-
-
450.7
17.3
138.7
2,4-di-F-Ph-
12.6
7.6
46.6
a
7
8
In vitro kinase assays were performed with the indicated purified recombinant MET or VEGFR-2
kinase domains (nM); b IC50 values (nM) for HGF-mediated autophosphoylation in MKN-45 cells; c Not
9
tested.
10
The initial SAR study of block A was performed (Table 1). Compound 13a bearing the
11
same block A (6,7-dimethoxyquinolin-4-yl) with 2 (Cabozantinib) only displayed moderate
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MET inhibitory activity (IC50 of 132.5 nM, 17.4-fold lower potency compared with 3). The
replacement of the quinoline with quinazoline (13b), thieno[3,2-b]pyridine (13c),
thieno[3,2-d]pyrimidine (13d), and imidazo[4,5-b]pyridin (13e), also resulted in a significant
loss of potency (IC50 of 151.9 nM to 664.2 nM, 20-fold to 87.4-fold lower potency compared
with 3). Interestingly, compound 13f bearing the same block A (2-amino-3-chloro-pyridin-
4-yl) with 3 displayed comparable MET inhibitory activity (IC50 of 9.9 nM) to that of
compound 3. The replacement of the 3-chlorine atom with iodine atom provided equivalent
potency (13g, IC50 of 10.7 nM), while the 3-amine-substituted analog 13h was less potent (IC50
of 103.0 nM).
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9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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More importantly, 13f and 13g displayed comparable cell-based MET inhibitory activity
(IC50 of 69 nM and 116.7 nM) and VEGFR-2 enzymatic inhibitory activity (IC50 of 279.9
nM and 63.2 nM) to that of compound 3 (IC50 of 46.6 nM and 138.7 nM). These results
indicated that our proposed scaffold hopping strategy was effective to provide novel MET
inhibitor.
Subsequently, a methyl group was introduced onto the C(3) position of 2,7-naphthyridine
in lead compound 13f. The resulting compound 19a (IC50 of 1.5 nM) exhibited a 6.6-fold
increase in potency compared with 13f and a 5.1-fold increase relative to 3. Compound 19b,
bearing 2-amino-3-iodo-pyridin-4-yl group in block A, also displayed excellent inhibitory
activity (IC50 of 2.4 nM). Meanwhile, 19a and 19b also exhibited comparable cell-based MET
inhibitory activity (IC50 of 89.8 nM and 117.8 nM) and VEGFR-2 enzymatic inhibitory
activity (IC50 of 261.1 nM and 102.6 nM) to that of compound 3.
Further SAR validation of block D indicated that the replacement of 4-F atom with 4-H
(19c, 19g), 2,4-di-F (19d, 19h) and 4-Cl (19e) resulted in the obvious reduction of MET
activity (IC50 of 18.0 nM and 33.2 nM, 3.7-fold to 22.1-fold less potent) compared with 19a,
while 19f (4-OCF3-phenyl group in block D) displayed no inhibitor activity in 1000 nM
(Table 1). Moreover, compounds 19c, 19e and 19h showed more potent VEGFR-2 inhibitory
activity (IC50 of 17.3 nM to 44.9 nM) to that of compound 3.
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Figure 4. (A)The proposed binding mode of 13f with MET; (B) The binding mode overlay of 13f
(magenta) and 19a (cyan) with MET. The yellow and green dashed lines represent H-bonds between
residues with 13f and 19a.
Molecular docking was further performed to understand the SAR of block C. As shown in
Figure 4A, the entire molecule of 13f was favorably located in the MET pocket as expected.
Due to the conservation of the key H-bond interactions between the carbonyl group in block
C with residues Asp1222, and the amino group in block A with the Met1160, 13f displayed
comparable MET enzymatic activity to that of 3. The introduction of a C(3) methyl group in
block C resulted in a conformational change-induced improvement of the H-bond interactions
between 19a and MET. As shown in Figure 4B, block C in 19a assumed an approximately 20°
transition compared to 13f, which led to an additional H-bond between the C(8) N-H group of
2,7-naphthyridine and the side-chain of the Asp1222 residue. As a result, 19a showed
6.6-fold improved potency over 13f.
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Table 2. Preliminary results of kinase profile of 13f
Inhibitory rate
Inhibitory rate
kinase
kinase
(% in 1 µM)
(% in 1 µM)
Aurora-B
Axl
44
97
Lck
Lyn
75
43
DDR2
DYRK2
EphA3
EphB4
Fes
98
-9
Mer
100
43
PKA
Ret
38
10
10
62
95
100
88
96
Ron
100
87
Ros
Fgr
Rse
83
Flt1
Src(1-530)
TrkA
TrkB
35
Flt4
99
Fms
100
2
3
4
5
6
7
8
The inhibitory activity of compound 13f against a panel of twenty two other kinases was
also assayed (Table 2). In contrast to its high potency against MET (IC50 = 9.9 nM), 13f also
exhibited high inhibitory effects against Axl, DDR2, Flt1, Flt4, Mer, Ret, Ron, TrkA and
TrkB (inhibitory rate > 90% in 1 µM). As expected, these data suggested that compound 13f
is a promising multitarget kinase inhibitor. More importantly, it provided a novel scaffold for
further selectivity enhancement.
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Table 3. In Vivo PK Profiles of Selected Compounds in Rata,b
AUC0-∞
Dose
Cmax
Tmax
T1/2
CL
Vz
Compd.
route
(µg* h
/mL)
6.7
F (%)
(mg/kg) (µg/mL)
(L/h/kg) (L/kg)
p.o.
i.v.
10
2.5
10
1.6
-
1.2
-
5.1
3.2
11.0
7.0
4.9
4.2
-
0.8
-
-
3.6
-
54
-
13f
3.1
p.o.
i.v.
3.7
-
2.3
-
21.2
10.5
25.7
13.1
-
19a
2.5
10
6.2
-
2.4
-
51
-
p.o.
i.v.
2.7
-
1.5
-
19b
2.5
0.4
2.9
49
a
10
11
12
Vehicle: 70% PEG400-30% water. Cmax, maximum concentration; Tmax, time of maximum concentration;
T1/2, half-lif; AUC0-∞, area under the plasma concentration time curve; CL, clearance; VZ, volume of
distribution; F, oral bioavailability. b Data reported as the average of six animals.
13
14
15
16
17
Three potent lead compounds 13f, 19a and 19b were selected for further evaluation of PK
properties in rats. As summarized in Table 3, compound 13f displayed favorable overall PK
profiles, with maximal plasma concentration (Cmax = 1.6 µg/mL), plasma exposure (AUC0-∞
= 6.7 µg·h/m), and oral bioavailability (F = 54%) after oral dose of 10 mg/kg . When the C(3)
methyl group was introduced into the 2,7-naphthyridine ring of block, the resulted
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compounds 19a and 19b exhibited higher Cmax (3.7 and 2.7 µg/mL) and AUC0-∞ (21.2 and
25.7µg·h/mL), and comparable oral bioavailability (F of 51% and 49%) at the same dose.
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4
Table 4. Plasma Protein Binding Data of Compound 13f in Rat, Mouse and Human (2 µM)
Free Fraction %
Compd.
Rat
Mouse
< 0.5
Hunan
< 0.5
13f
< 0.5
5
6
The plasma protein binding data of compound 13f in three different species is listed in
Table 4. As the buffer side response of 13f is lower than five thousandths of the dose, the
plasma protein binding law of 13f is greater than 99.5%. As far as we know, significant
plasma protein binding is common for highly lipophilic compounds.
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Table 5. In vivo Tumor Growth Inhibition Activity
Tumor
model
Dose a
Compd.
TGI (%)b
PRc
(mg/kg)
12.5
25
50
25
50
25
40
25
25
53*
100**
114**
50**
95**
44
0/6
3/6
6/6
0/6
0/6
0/6
0/6
2/6
0/6
U-87 MG
13f
HT-29
19a
3
U-87 MG
72
U-87 MG
HT-29
98**
15
12
13
a 1Q.D.×21/70% PEG-400/H2O/p.o.; b TGI, Tumor growth inhibition value; *: P<0.05, **: P<0.01;
c PR, partial regression.
14
15
16
17
18
19
20
21
The in vivo antitumor efficacy of 13f and 19a were further evaluated in the U-87 MG
human glioblastoma xenograft model20 (Table 5 & Figure 5A). When administered orally
Q.D., two compounds 13f, 19a both induced dose-dependent tumor growth inhibition, with
the minimum effective doses (MED/ED50, 50% inhibition of tumor growth) of ~12.5, ~32.5
mg/kg, respectively. After dose at 25 mg/kg, 13f displayed a comparable in vivo efficacy
(Tumor Growth Inhibition/TGI of 100%) to that of compound 3 (TGI of 98%). Partial tumor
regressions of all animals (PR 6/6, TGI of 114%) were observed at higher doses of 13f (50
mg/kg).
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Figure 5. Antitumor efficacy of 13f in the U-87 MG (A) and HT-29 (B) xenograft models. Tumor-bearing
nude mice were randomly divided into groups when the tumor volume reached 100-200 mm3 and given
13f p.o. at the indicated dose levels or vehicle alone over the designated treatment schedule. Data are
presented as the mean (+ SEM; n = 6 mice per group).
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Compound 13f were further evaluated in the HT-29 human colorectal adenocarcinoma
xenograft model (Table 5 & Figure 5B). In the HT-29 model, 13f displayed better in vivo
efficacy to that of 3 (TGI of 50 Vs 15 % inhibition at 25 mg/kg). Moreover, a significant
inhibitory effect (TGI of 95%) on tumor growth was observed at a higher dose (50 mg/kg).
Taken together, 13f displayed excellent in vivo efficacy both in the U-87 MG and HT-29
models.
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4. Conclusions
In the present work, we described the design, synthesis, and biological evaluation of a
series of conformationally constrained 2,7-naphthyridone-based derivatives of
BMS-777607 as new MET inhibitors. Extensive SAR studies led to the identification of
13f, which displayed comparable MET inhibitory activity (IC50 = 9.9 nM and 69 nM) to
that of BMS-777607 (IC50 = 7.6 nM and 46.6 nM) at both the enzyme-based and cell-based
assay. More importantly, 13f exhibited favorable oral bioavailability (54% in rat) and
significant in vivo efficacy (TGI of 114 % and 95 % in 50 mg/kg, respectively) both in the
U-87 MG and HT-29 xenograft models. The favorable drug-likeness of 13f indicated that
2,7-naphthyridinone may be used a promising novel scaffold for antitumor drug
development. Nowadays, 13f has been advanced into preclinical studies.
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5. Experimental
5.1. General methods
3
Unless otherwise noted, all chemical reagents were commercially available and treated
with standard methods. Silica gel column chromatography (CC). silica gel (200-400 Mesh;
Qingdao Makall Group Co., Ltd; Qingdao; China). Solvents were dried in a routine way
and redistilled. All reactions involving air- or moisture-sensitive reagents were performed
under a nitrogen or argon atmosphere. Melting points of compounds were measured on a
Melt-Temp II apparatus and uncorrected. 1H NMR spectra (400 MHz ) and 13C NMR (100
MHz) spectra were recorded on a Bruker BioSpin AG (Ultrashield Plus AV 400)
spectrometer as deuterochloroform (CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6) solutions
using tetramethylsilane (TMS) as an internal standard (δ = 0) unless noted otherwise. MS
spectra were obtained on an Agilent technologies 6120 quadrupole LC/MS (ESI).
High-resolution mass spectra (HR-MS) were obtained on an Agilent 6224 TOF LC/MS
(USA) All reactions were monitored using thin-layer chromatography (TLC) on silica gel
plates. Yields were of purified compounds and were not optimized.
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5.2. General procedures for the synthesis of intermediates
5.2.1 1-(4-fluorophenyl)-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (10).
A dried flask was charged with N,N'-dimethyl-1,2-ethanediamine (14.0 mmol), K3PO4
(14.0mmol), 4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile 6 (12.7 mmol), CuI (14.0
mmol) and anhydrous dioxane (50 mL) under nitrogen. The mixture was heated to 100 °C
and stirred for 12 h, then cooled to room temperature and filtered. The filtrate was
concentrated under reduced pressure, and purified by chromatography (CH2Cl2/MeOH =
100:1) to yield 10 as a yellow solid (41%). 1H-NMR (400 M, CDCl3) δ 7.44 (d, J = 7.2 Hz,
1H), 7.33-7.36 (m, 2H), 7.17-7.21 (m, 2H), 6.24 (d, J = 7.2 Hz, 1H), 2.51 (s, 3H). MS
(ESI). 228.1 (M)+.
5.2.2 (E)-4-(2-(dimethylamino)vinyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-
Carbonitrile (11).
A dried flask was charged with N,N-dimethylformamide dimethyl acetal (10 mmol), 10
(10 mmol), and anhydrous DMF (50 mL) under nitrogen. The mixture was heated to 90 °C
and stirred for 2 h. The resulted solution was concentrated under reduced pressure, and
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purified by chromatography (CH2Cl2/MeOH = 50:1) to yield 11 as a yellow solid (93%).
1H-NMR (400 M, CDCl3) δ 7.28-7.35 (m, 3H), 7.11-7.16 (m, 3H), 6.23 (d, J = 7.6 Hz, 1H),
5.42 (d, J = 13.2 Hz, 1H), 2.51 (s, 3H). MS (ESI). 283.1 (M)+.
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4
5.2.3 2-(4-fluorophenyl)-2,7-naphthyridine-1,8(2H,7H)-dione (12).
5
A solution of 11 (3 mmol) in H2SO4 (15 mL) was heated to 110 °C under nitrogen for 2
h. Ice-cold water was added, and the mixture was extracted with dichloromethane. The
organic layer was dried and concentrated. The residue was purified by chromatography
(CH2Cl2/MeOH = 10:1) to yield 12 as a yellow solid (78%). 1H-NMR (400 M, DMSO-d6)
δ 11.39 (bs, 1H), 7.69 (d, J = 7.2 Hz, 1H), 7.31-7.48 (m, 5H), 6.31-6.40 (m, 2H). MS (ESI).
256.1 (M)+.
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5.2.4 8-chloro-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one (4).
A solution of 12 (2 mmol) in POCl3 (25 mL) was heated to 110 °C under nitrogen for 1
h. Ice-cold water was added, and the mixture was extracted with dichloromethane. The
organic layer was dried and concentrated. The residue was purified by chromatography
1
(CH2Cl2/MeOH = 50:1) to yield 4 as a yellow solid (85%). H-NMR (400 M, CDCl3) δ
8.50 (d, J = 4.2 Hz, 1H), 7.77 (d, J = 7.2 Hz, 1H), 7.67 (d, J = 4.2 Hz, 1H), 7.52-7.56 (m,
2H), 7.37-7.41 (m, 2H), 6.73 (d, J = 7.2 Hz, 1H). MS (ESI). 274.0 (M)+.
5.2.5 1-(4-fluorophenyl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (15a).
A solution of 14 (5 mmol), pentane-2,4-dione (5 mmol) and piperidine in EtOH (50 mL)
was heated to 90 °C under nitrogen for 2 h, then cooled to room temperature and filtered.
The filtrate was concentrated under reduced pressure to yield 15a as a white solid (90%).
1H-NMR (400 M, CDCl3) δ 7.16-7.26 (m, 4H), 6.14 (s, 1H), 2.45 (s, 3H), 2.03 (s, 3H). MS
(ESI). 242.0 (M)+.
5.2.6 (E)-4-(2-(dimethylamino)vinyl)-1-(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydro-
pyridine-3-carbonitrile (16a).
Prepared according to the procedure for the preparation of 11, from 15a, to yield 16a as
yellow solid (75%). 1H-NMR (400 M, CDCl3) δ 7.16-7.30 (m, 5H), 6.12 (s, 1H), 5.37 (d, J
= 13.2 Hz, 1H), 2.95 (s, 6H), 1.93 (s, 3H). MS (ESI). 297.1 (M)+.
5.2.7 2-(4-fluorophenyl)-3-methyl-2,7-naphthyridine-1,8(2H,7H)-dione (17a).
Prepared according to the procedure for the preparation of 12, from 16a, to yield 17a as
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yellow solid (80%). H-NMR (400 M, DMSO-d6) δ 11.23 (bs, 1H), 7.34-7.43 (m, 5H),
6.32 (s, 1H), 6.18 (bs, 1H), 1.90 (s, 3H). MS (ESI). 270.0 (M)+.
3
5.2.8 8-chloro-2-(4-fluorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (18a).
Prepared according to the procedure for the preparation of 4, from 17a, to yield 18a as
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yellow solid (84%). H-NMR (400 M, CDCl3) δ 8.39 (d, J = 5.6 Hz, 1H), 7.20-7.31 (m,
6
4H), 6.36 (s, 1H), 2.08 (s, 3H). MS (ESI). 288.0 (M)+.
7
5.2.9 8-chloro-3-methyl-2-phenyl-2,7-naphthyridin-1(2H)-one (18b).
Prepared according to the procedure for the preparation of 18a, from
2-cyano-N-phenylacetamide (14b), to yield 18b as yellow solid. 1H-NMR (400 M, CDCl3)
δ 8.39 (d, J = 5.2 Hz, 1H), 7.46-7.56 (m, 3H), 7.20-7.28 (m, 3H), 6.34 (s, 1H), 2.03 (s, 3H).
MS (ESI). 270.0 (M)+.
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5.2.10 8-chloro-2-(2,4-difluorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (18c).
Prepared according to the procedure for the preparation of 18a, from
1
2-cyano-N-(2,4-difluorophenyl)acetamide (14c), to yield 18c as yellow solid. H-NMR
(400 M, CDCl3) δ 8.41 (d, J = 5.6 Hz, 1H), 7.21-7.29 (m, 3H), 7.02-7.08 (m, 3H), 6.39 (s,
1H), 2.07 (s, 3H). MS (ESI). 306.0 (M)+.
5.2.11 8-chloro-2-(4-chlorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (18d).
Prepared according to the procedure for the preparation of 18a, from
N-(4-chlorophenyl)-2-cyanoacetamide (14d), to yield 18d as yellow solid. 1H-NMR (400 M,
CDCl3) δ 8.40 (d, J = 5.2 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.17-7.26 (m, 3H), 6.37 (s, 1H),
2.04 (s, 3H). MS (ESI). 304.0 (M)+.
5.2.12
8-chloro-3-methyl-2-(4-(trifluoromethoxy)phenyl)-2,7-naphthyridin-1(2H)-one
(18e).
Prepared according to the procedure for the preparation of 18a, from
2-cyano-N-(4-(trifluoromethyl)phenyl)acetamide (14e), to yield 18e as yellow solid.
1H-NMR (400 M, CDCl3) δ 8.41 (d, J = 5.2 Hz, 1H), 7.21-7.38 (m, 5H), 6.37 (s, 1H), 2.05
(s, 3H). MS (ESI). 354.0 (M)+.
5.3. General procedures for the synthesis of targets 13a-13h and 19a-19h
5.3.1 8-((4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluorophenyl)-
2,7-naphthyridin-1(2H)-one (13a).
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A dried flask was charged with 4 (1 mmol), 1,3-Bis(diphenylphosphino)propane (20
mmol %), sodium tert-butoxide (1.2 mmol), Pd2(dba)3 (10 mmol %), 4-((6,7-dimethoxy-
quinolin-4-yl)oxy)-3-fluoroaniline 5a and anhydrous dioxane (15 mL) under nitrogen. The
mixture was heated to 100 °C and stirred for 3 h, then cooled to room temperature and
filtered. The filtrate was concentrated under reduced pressure, and purified by
chromatography (CH2Cl2 /MeOH = 20:1) to yield 8-((4-((6,7-dimethoxyquinolin-4-yl)oxy)
-3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one 13a as a yellow
3
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solid (55%). M.p. 255-256 °C. H-NMR (400 M, CDCl3) δ 12.11 (s, 1H), 8.37-8.40 (m,
9
2H), 7.83 (s, 1H), 7.52 (d, J = 6.8 Hz, 1H), 7.46 (d, J = 6.4 Hz, 1H), 7.39-7.42 (m, 2H),
7.30-7.33 (m, 2H), 7.24-7.28 (m, 2H), 6.87 (d, J = 4.4 Hz, 1H), 6.53 (d, J = 6.4 Hz, 1H),
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6.34-6.37 (m, 2H), 4.00 (s, 3H) , 3.73 (s, 3H); C-NMR (100 M, CDCl3) δ 177.3, 164.1,
163.3, 156.2, 155.6, 155.1, 153.4, 150.5, 147.3, 145.9, 142.0, 138.2, 136.2, 129.1, 128.6,
122.1, 121.0, 116.9, 116.7, 111.8, 110.8, 109.5, 108.4, 107.6, 106.2, 105.8, 98.2, 56.3, 56.0.
MS (ESI). 553.0 [M+H]+. HR-MS (EI) m/z calcd for C31H22F2N4O4, 552.1609; found
552.1638 [M]+.
5.3.2
8-((4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluoro-
phenyl)-2,7-naphthyridin-1(2H)-one (13b).
Prepared according to the procedure for the preparation of 13a, from
4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline 5b and 4, to yield 13b as yellow
solid (61%), M.p. 199-200 °C. 1H-NMR (400 M, CDCl3) δ 11.86 (s, 1H), 8.64 (s, 1H), 8.34
(d, J = 4.4 Hz, 1H), 8.25 (d, J = 6.4 Hz, 1H), 7.59 (s, 1H), 7.40-7.46 (m, 4H), 7.23-7.29 (m,
13
4H), 6.76 (d, J = 4.4 Hz, 1H), 6.48 (d, J = 6.4 Hz, 1H), 4.08 (s, 6H); C-NMR (100 M,
DMSO-d6) δ 182.1, 165.0, 163.3, 157.1, 156.6, 155.1, 153.8, 150.9, 150.2, 149.0, 146.5,
138.3, 136.1, 130.5, 130.0, 128.9, 125.1, 117.2, 116.8, 110.8, 109.1, 108.7, 107.9, 106.7,
105.8, 100.8, 56.6. MS (ESI). 553.9 [M+H]+ HR-MS (EI) m/z calcd for C30H21F2N5O4,
.
553.1562; found 554.1640 [M+H]+.
5.3.3 8-((3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)phenyl)amino)-2-(4-fluorophenyl)-2,7-
naphthyridin-1(2H)-one (13c).
Prepared according to the procedure for the preparation of 13a, from
3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline 5c and 4, to yield 13c as yellow solid
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(50%), M.p. 125-126 °C. H-NMR (400 M, CDCl3) δ 11.93 (s, 1H), 8.58 (d, J = 5.6 Hz,
1H), 8.38-8.34 (m, 3H),7.43-7.21 (m, 7H), 7.09 (t, J = 9.2 Hz, 1H), 6.81 (d, J = 5.4Hz, 1H),
6.52 (d, J =5.6 Hz, 1H), 6.49 (d, J =5.4 Hz, 1H); 13C-NMR (100 M, CDCl3) δ 163.1, 162.9,
161.2, 160.4, 158.8, 156.3, 155.6, 150.6, 150.1, 145.8, 139.2, 136.3, 136.0, 130.7, 128.7,
125.2, 123.4, 122.0, 116.8, 116.6, 116.4, 110.3, 109.4, 106.5, 106.1, 103.1. MS (ESI).
498.8 [M+H]+. HR-MS (EI) m/z calcd for C27H16F2N4O2S, 498.0962; found 499.1045
[M+H]+.
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5.3.4 8-((3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)amino)-2-(4-fluorophenyl)-
2,7-naphthyridin-1(2H)-one (13d).
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Prepared according to the procedure for the preparation of 13a, from
3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)aniline 5d and 4, to yield 13d as yellow solid
(66%), M.p. 155-156°C. 1H-NMR (400 M, CDCl3) δ 11.86 (s, 1H), 8.72 (s, 1H), 8.34 (d, J
= 5.4 Hz, 1H), 8.23 (d, J =10.5 Hz, 1H), 7.97 (d, J = 5.4Hz, 1H), 7.50 (d, J = 5.5Hz, 1H),
13
7.42-7.21 (m, 7H), 6.77 (d, J = 5.4Hz, 1H), 6.48 (d, J =7.4 Hz, 1H); C-NMR (100 M,
CDCl3) δ 163.2, 161.2, 160.4, 156.3, 155.4, 154.4, 152.3, 150.7, 145.8, 139.4, 139.3, 136.4,
135.9, 135.2, 133.7, 128.7, 123.4, 117.3, 116.8, 116.6, 116.1, 110.1, 109.1, 108.9, 106.5,
106.1. MS (ESI). 499.8 [M+H]+. HR-MS (EI) m/z calcd for C26H15F2N5O2S, 499.0915;
found 500.1002 [M+H]+.
5.3.5 8-((3-fluoro-4-((2-hydroxy-3H-imidazo[4,5-b]pyridin-7-yl)oxy)phenyl)amino)-2-(4-
fluorophenyl)-2,7-naphthyridin-1(2H)-one (13e).
Prepared according to the procedure for the preparation of 13a, from
7-(4-amino-2-fluorophenoxy)-3H-imidazo[4,5-b]pyridin-2-ol 5e and 4, to yield 13e as
yellow solid (62%),, M.p. 222-223 °C. 1H-NMR (400 M, DMSO-d6) δ 11.98 (s, 1H), 11.37
(s, 1H), 11.23 (s, 1H), 8.35 (d, J = 5.4 Hz, 1H), 8.29 (d, J = 11.2 Hz, 1H), 7.76 (d, J = 6.0
Hz, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.60-7.57 (m, 2H), 7.47-7.39 (m, 3H), 7.30 (t, J = 9.0
Hz, 1H), 7.03 (d, J = 5.4Hz, 1H), 6.71 (d, J = 7.3 Hz, 1H), 6.33 (d, J =6.0 Hz, 1H);
13C-NMR (100 M, DMSO-d6) δ 162.8, 162.6, 160.2, 155.4, 154.5, 154.2, 150.0, 146.8,
145.7, 141.4, 138.5, 138.4, 137.8, 136.6, 129.5, 123.1, 116.2, 115.9, 112.1, 110.8, 108.3,
105.8, 105.2, 104.0. MS (ESI). 499.1 [M+H]+; HR-MS (EI) m/z calcd for C26H16F2N6O3,.
498.1252; found 521.1141 [M+Na]+.
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5.3.6 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluoro-
phenyl)-2,7-naphthyridin-1(2H)-one (13f).
3
A dried flask was charged with 4 (1 mmol), 1,3-Bis(diphenylphosphino)propane (20
mmol %), sodium tert-butoxide (1.2 mmol), Pd2(dba)3 (10 mmol %), 4-(4-amino-2-
fluorophenoxy)-3-chloropicolinonitrile 5f and anhydrous dioxane (15 mL) under nitrogen.
The mixture was heated to 100 °C and stirred for 3 h, then cooled to room temperature and
filtered. The filtrate was concentrated under reduced pressure, and purified by
chromatography (CH2Cl2/MeOH = 20:1) to yield 3-chloro-4-(2-fluoro-4-((7-(4-fluoro
phenyl)-8-oxo-7,8-dihydro-2,7-naphthyridin-1-yl)amino)phenoxy)picolinonitrile as a
yellow solid (80%). A solution of above nitrile derivative (1 mmol) and 2,2,6,6-tetramethyl
piperidin-1-ol (5 mmol) in CH2Cl2 (10 mL)was heated to 40 °C for 5 h. The resulted
solution was concentrated under reduced pressure, and purified by chromatography
(CH2Cl2/MeOH =30:1) to yield 3-chloro-4-(2-fluoro-4-((7-(4-fluoro-phenyl)-8-oxo-
7,8-dihydro-2,7-naphthyridin-1-yl)amino)phenoxy)picolinamide as a yellow solid (63%). A
solution of above carboxy amide derivative (1 mmol) and (diacetoxyiodo)benzene (1.1
mmol) in acetonitrile (10 mL) was stired at 0 °C for 1 h. The resulted solution was
concentrated under reduced pressure, and purified by chromatography (CH2Cl2/MeOH =
20:1) to yield 13f as a yellow solid (78%), M.p. 244-245 °C. 1H-NMR (400 M, DMSO-d6)
δ 11.83 (s, 1H), 8.34 (d, J = 4.4 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.78 (bs, 1H), 7.37-7.41
(m, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.23-7.28 (m, 4H), 7.09 (t, J = 7.2 Hz, 1H), 6.77 (d, J =
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4.4 Hz, 1H), 6.48 (d, J = 4.8 Hz, 1H), 5.04 (s, 2H); C-NMR (100 M, CDCl3) δ 164.0,
163.2, 162.5, 162.0, 156.5, 156.0, 155.3, 152.9, 150.6, 146.7, 145.8, 139.0, 136.3, 135.4,
128.7, 123.1, 116.9, 115.3, 110.2, 109.1, 106.5, 106.0, 102.4. MS (ESI). 491.8 [M+H]+.
HR-MS (EI) m/z calcd for C30H26FN5O2, 491.0961; found 492.1029 [M+H]+.
5.3.7 8-((4-((2-amino-3-iodopyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluorophenyl)-
2,7-naphthyridin-1(2H)-one (13g).
Prepared according to the procedure for the preparation of 13f, from
4-(4-amino-2-fluorophenoxy)-3-iodopicolinonitrile 5g and 4, to yield 13g as yellow solid
1
(33%), M.p. 251-252 °C. H-NMR (400 M, DMSO-d6) δ 11.91 (s, 1H), 8.47 (s, 1H), 8.19
(d, J = 11.2 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H), 7.63-7.59 (m, 2H),
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7.45-7.41 (m, 3H), 7.26 (t, J = 9.2 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.22 (s, 2H), 5.80 (d, J
13
= 5.6 Hz, 1H); C-NMR (100 M, DMSO-d6) δ 164.2, 163.0, 162.6, 161.8, 161.3, 154.7,
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152.4, 149.7, 148.9, 143.1, 140.1, 138.5, 136.7, 135.9, 129.9, 123.8, 117.0, 116.7, 116.5,
115.3, 109.1, 108.8, 107.0, 101.4, 100.1. MS (ESI). 583.0 [M]+. HR-MS (EI) m/z calcd for
C25H16F2IN5O2 , 583.0317; found 584.0396 [M+H]+.
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6
5.3.8 8-((4-((2,3-diaminopyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-
naphthyridin-1(2H)-one (13h).
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8
Prepared according to the procedure for the preparation of 13f, from
3-amino-4-(4-amino-2-fluorophenoxy)picolinonitrile 5h and 4, to yield 13h as yellow solid
(36%), M.p. 230-231 °C. 1H-NMR (400 M, DMSO-d6) δ 11.91 (s, 1H), 8.33 (d, J = 5.4 Hz,
1H), 8.23 (d, J = 11.2 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.61-7.56 (m, 2H), 7.43-7.37 (m,
3H), 7.21 (d, J = 5.7 Hz, 1H), 7.10 (t, J = 9.1 Hz, 1H), 7.01 (d, J = 5.4 Hz, 1H), 6.70 (d, J =
7.4 Hz, 1H), 5.90 ( d, J = 5.6 Hz, 1H), 5.55 (s, 2H), 4.48 (s, 2H) ; 13C-NMR (100 M,
DMSO-d6) δ 162.5, 160.3, 155.4, 154.3, 151.9, 150.2, 149.9, 148.1, 145.7, 137.6, 137.2,
136.5, 136.3, 135.6, 129.4, 122.2, 118.3, 116.1, 116.0, 115.8, 110.4, 108.1, 105.6, 105.1,
101.5. MS (ESI). 473.1 [M+H]+. HR-MS (EI) m/z calcd for C25H18F2N6O2, 472.1459;
found 473.1541 [M+H]+.
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5.3.9 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluoro-
phenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (19a).
Prepared according to the procedure for the preparation of 13f, from
8-chloro-2-(4-fluorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one 18a and 5f, to yield 19a
1
as yellow solid (35%), M.p. 213-214 °C. H-NMR (400 M, DMSO-d6) δ 11.90 (s, 1H),
8.25-8.30 (m, 2H), 7.74 (d, J = 4.4 Hz, 1H), 7.47-7.50 (m, 3H), 7.40-7.43 (m, 2H),7.26 (t, J
= 7.2 Hz, 1H), 6.91 (d, J = 4.4 Hz, 1H), 6.65 (s, 1H), 6.35 (s, 2H), 5.92 (d, J = 4.4 Hz, 1H),
13
1.99 (s, 3H); C-NMR (100 M, CDCl3) δ 163.7, 163.1, 160.6, 159.9, 157.4, 155.2, 154.4,
152.0, 149.7, 145.5, 145.2, 134.4, 134.2, 130.8, 123.3, 116.5, 116.0, 110.1, 108.1, 106.5,
104.8, 104.4, 100.1, 21.2. MS (ESI). 506.1 [M+H]+. HR-MS (EI) m/z calcd for
C26H18ClF2N5O2, 505.1117; found 506.1184 [M+H]+.
5.3.10 8-((4-((2-amino-3-iodopyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-fluorophenyl)-
3-methyl-2,7-naphthyridin-1(2H)-one (19b).
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Prepared according to the procedure for the preparation of 13f, from
8-chloro-2-(4-fluorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one 18a and 5g, to yield 19b
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3
as yellow solid (35%), M.p. 223-224 °C. H-NMR (400 M, DMSO-d6) δ 11.90 (s, 1H),
4
8.30 (d, J = 5.5 Hz, 1H), 8.26 (d, J = 11.2 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H), 7.50-7.40 (m,
5H), 7.22 (t, J = 9.1 Hz, 1H), 6.91 (d, J = 5.5 Hz, 1H), 6.65 (s, 1H), 6.16 (s, 2H), 5.80 (d, J
5
13
6
= 5.5 Hz, 1H), 1.99 (s, 3H); C-NMR (100 M, DMSO-d6) δ 164.1, 163.7, 160.9, 160.1,
7
157.6, 155.3, 154.5, 149.8, 149.3, 145.5, 145.3, 134.5, 134.3, 130.8, 130.7, 123.3, 116.5,
116.3, 116.0, 110.0, 108.2, 106.5, 104.8, 104.4, 100.2, 21.3. MS (ESI). 598.0 [M+H]+.
HR-MS (EI) m/z calcd for C26H18F2IN5O2, 597.0473; found 598.0561 [M+H]+.
5.3.11 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-methyl-2-
phenyl-2,7-naphthyridin-1(2H)-one (19c).
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Prepared according to the procedure for the preparation of 13f, from
8-chloro-3-methyl-2-phenyl-2,7-naphthyridin-1(2H)-one 18b and 5f, to yield 19c as yellow
1
solid (62%), M.p. 201-202 °C. H-NMR (600 M, CDCl3) δ 11.78 (s, 1H), 8.28 (d, J = 5.4
Hz, 1H), 8.18 (d, J = 12.0 Hz, J2=3.6 Hz, 1H), 7.77 (d, J = 6.0 Hz, 1H), 7.61-7.27 (m, 4H),
7.06 (t, J = 9.1 Hz, 1H), 6.70 (t, J = 4.8 Hz, 1H), 6.37 (s, 1H), 6.07 (d, J = 6.0 Hz, 1H),
5.31 (d, J = 5.4 Hz, 1H), 4.94 (s, 2H), 2.03 (s, 3H); 13C-NMR (100 M, DMSO-d6) δ 163.6,
159.9, 157.4, 155.2, 154.4, 152.0, 147.2, 145.4, 145.2, 138.9, 138.8, 138.0, 134.4, 134.2,
129.6, 128.8, 128.5, 123.3, 116.0, 110.0, 108.0, 107.8, 104.7, 104.4, 100.0, 21.3. MS (ESI).
488.0 [M+H]+. HR-MS (EI) m/z calcd for C26H19ClFN5O2, 487.1211; found 487.1285
[M]+.
5.3.12 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(2,4-difluoro-
phenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (19d).
Prepared according to the procedure for the preparation of 13f, from 8-chloro-2-(2,4-
difluorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one 18c and 5f, to yield 19d as yellow
1
solid (47%), M.p. 210-211 °C. H-NMR (400 M, DMSO-d6) δ 11.73 (s, 1H), 8.33 (d, J =
5.2 Hz, 1H), 8.27 (d, J = 11.2 Hz, 1H), 7.75 (d, J = 5.6 Hz, 1H), 7.71-7.27 (m, 4H), 7.30 (t,
J = 8.8 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 6.71 (s, 1H), 6.44 (s, 2H), 5.92 (d, J = 5.6 Hz,
1H), 2.03 (s, 3H); 13C-NMR (100 M, DMSO-d6) δ 161.6, 161.4, 161.3, 161.2, 157.4,157.3,
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155.2, 151.0, 121.3, 116.1, 106.1, 106.0, 102.8, 102.2, 22.5. MS (ESI). 524.1[M+H]+.
HR-MS (EI) m/z calcd for C26H17ClF3N5O2, 523.1023; found 524.1108 [M+H]+.
5.3.13 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(4-chloro-
phenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (19e).
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Prepared according to the procedure for the preparation of 13f, from 8-chloro-2-(4-
chlorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one 18d and 5f, to yield 19e as yellow
6
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solid (57%), M.p. 221-222 °C. H-NMR (400 M, CDCl3) δ 11.70 (s, 1H), 8.28 (d, J = 5.2
8
Hz, 1H), 8.18 (d, J = 10.8 Hz, 1H), 7.77 (d, J = 5.6 Hz, 1H), 7.58-7.20 (m, 5H), 7.07 (t, J =
8.8 Hz, 1H), 6.69 (d, J = 5.2 Hz, 1H), 6.37 (s, 1H), 6.06 (m, 1H), 4.97 (s, 2H), 2.19 (s, 3H);
13C-NMR (100 M, DMSO-d6) δ 163.6, 159.8, 157.4, 155.2, 154.4, 152.0, 149.8, 147.2,
145.2, 138.8, 138.7, 136.9, 134.4, 134.3, 133.5, 130.6, 129.6, 123.3, 116.0, 110.1, 108.1,
107.8, 104.9, 104.3, 100.0, 21.2. MS (ESI). 522.0 [M+H]+. HR-MS (EI) m/z calcd for
C26H18Cl2FN5O2, 521.0822; found 522.0891 [M+H]+.
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5.3.14 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-methyl-2-(4-
(trifluoromethoxy)phenyl)-2,7-naphthyridin-1(2H)-one (19f).
Prepared according to the procedure for the preparation of 13f, from 8-chloro-3-methyl-
2-(4-(trifluoromethoxy)-phenyl)-2,7-naphthyridin-1(2H)-one 18e and 5f, to yield 19f as
yellow solid (39%), M.p. 233-234 °C. 1H-NMR (400 M, CDCl3) δ 11.67 (s, 1H), 8.29 (d, J
= 5.2 Hz, 1H), 8.18 (d, J = 10.8 Hz, 1H), 7.78 (d, J = 5.2 Hz, 1H), 7.43-7.07 (m, 6H), 6.70
(d, J = 5.2 Hz, 1H), 6.38 (d, J = 5.2 Hz, 1H), 6.06 (s, 1H), 4.95 (s, 2H), 2.04 (s, 3H);
13C-NMR (100 M, DMSO-d6) δ 163.6, 159.8, 157.4, 155.2, 154.4, 152.0, 149.8, 148.2,
147.2, 145.2, 138.8, 137.0, 134.4, 130.8, 123.3, 122.2, 121.3, 118.7, 116.0, 110.2, 108.1,
107.9, 104.9, 104.3, 100.8, 100.0, 21.2. MS (ESI). 572.0 [M+H]+. HR-MS (EI) m/z calcd
for C27H18ClF4N5O3, 571.1034; found 572.1109 [M+H]+.
5.3.15 8-((4-((2-amino-3-iodopyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-methyl-2-phenyl-
2,7-naphthyridin-1(2H)-one (19g).
Prepared according to the procedure for the preparation of 13f, from 18b and 5g, to
yield 19g as yellow solid (56%), M.p. 218-219 °C. 1H-NMR (400 M, DMSO-d6) δ 11.96 (s,
1H), 8.30 (d, J = 5.6 Hz, 1H), 8.25 (d, J = 11.2 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H), 7.62-7.40
(m, 6H), 7.24 (t, J = 9.1 Hz, 1H), 6.92 (d, J = 5.6 Hz, 1H), 6.66 (s, 1H), 6.32 (s, 2H), 5.81
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(d, J = 5.6 Hz, 1H), 1.98 (s, 3H); 13C-NMR (100 M, DMSO-d6) δ 164.6, 163.6, 160.2,
155.2, 154.3, 149.7, 148.0, 145.5, 145.2, 138.9, 138.8, 138.0, 134.5, 129.6, 128.8, 128.5,
123.3, 116.0, 110.1, 108.0, 107.8, 104.7, 104.4, 99.6, 21.3. MS (ESI). 58.0 [M+H]+.
HR-MS (EI) m/z calcd for C26H19FIN5O2, 579.0567; found 580.0643 [M+H]+.
5.3.16 8-((4-((2-amino-3-iodopyridin-4-yl)oxy)-3-fluorophenyl)amino)-2-(2,4-difluoro-
phenyl)-3-methyl-2,7-naphthyridin-1(2H)-one (19h).
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Prepared according to the procedure for the preparation of 13f, from 18c and 5g, to
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yield 19h as yellow solid (56%), M.p. 225-226 °C. H-NMR (400 M, DMSO-d6) δ 11.71
9
(s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 8.26 (d, J = 12.0 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H),
7.70-7.34 (m, 4H), 7.23 (t, J = 9.2 Hz, 1H), 6.93 (d, J = 5.2 Hz, 1H), 6.71 (s, 1H), 6.20 (s,
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2H), 5.78 (d, J = 5.6 Hz, 1H), 2.03 (s, 3H); C-NMR (100 M, DMSO-d6) δ 164.1, 163.2,
160.9, 155.2, 154.4, 151.9, 150.3, 149.3, 145.4, 145.1, 138.5, 135.0, 132.1, 123.3, 121.8,
116.2, 112.7, 112.5, 110.2, 108.3, 108.0, 105.3, 105.0, 103.9, 99.6, 20.6. MS (ESI). 616.0
[M+H]+. HR-MS (EI) m/z calcd for C26H19FIN5O2, 615.0379; found 616.0468 [M+H]+.
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5.4. Molecular docking.
The three dimensional (3D) structure of the MET kinase complex (PDB code: 3F82) and
VEGFR-2 kinase (PDB code: 3U6J) complex was obtained from PDB database. All water
molecules and ligand were removed from the complex structure and hydrogen atoms were
added with pH equaling to 7.0 using Sybyl-X. The AutoDock 4.225 program was applied to
docking compound 13f and 19a into the binding site of MET kinase or VEGFR-2 kinase.
The Gasteiger charges were used for these inhibitors. In the docking process, a
conformational search was performed for the ligand using the Solis and Wets local search
method, and the Lamarckian genetic algorithm (LGA)26,27 was applied for the
conformational search of the binding complex of ligand with two kinases. Among a series
of docking parameters, the grid size was set to be 70x70x80 in 3F82, 46x40x60 in 3U6J,
and the used grid space was the default value of 0.375 Å. Among a set of 100 candidates of
the docked complex structures, the best one was first selected according to the interaction
energy and was then compared with the conformation of ligand (3, BMS-777607) extracted
from the crystal structure.
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5.5. In Vivo Antitumor Activity Assay.
Female nude mice (4-6 weeks old) were housed and maintained under specific-pathogen
free conditions. Animal experiments were performed according to institutional ethical
guidelines of animal care. Tumor cells were inoculated into the flanks of athymic nude
mice (2×106 cells/mouse). When the tumor volume reached about 100-200 mm3, the mice
were randomly assigned into control and treatment groups. Control groups were given
vehicle alone, and treatment groups received synthesized compounds as indicated doses via
p.o. administration 7 days per week for 3 weeks. The sizes of the tumors were measured
three times per week using microcaliper. The tumour volume (V) was calculated as
follows: V =[length (mm) ×width2 (mm2)]/2. Percent (%) inhibition values (TGI) were
measured on the final day of study for drug treated compared with vehicle-treated mice and
were calculated as 100% × (1- ((treatedfinal day−treatedday 0)/controlfinal day −controlday 0)).
Significant differences between the treated versus the control groups (P ≤ 0.01) were
determined using Student’s.
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Acknowledgement
This work was supported by the National Key Research and Development Program of
China (No. 2017YFA0505200), the National Natural Science Foundation of China (No.
201502063), the Natural Science Foundation of Hubei Province (No. 2016CFB562) and the
self-determined research funds of CCNU from the colleges' basic research and operation of
MOE (No. CCNU18TS009).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at.
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Highlights:
2,7-Naphthyridinone-Based MET Kinase Inhibitors: A
Promising Novel Scaffold for Antitumor Drug Development
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2,7-Naphthyridinone was developed as a promising scaffold for the discovery of
new MET-targeted antitumor drug.
The 2,7-naphthyridone fragment was utilized to conformationally restrain key
pharmacophoric groups (block C) of BMS-777607 and resulted in the discovery
of a new potent MET inhibitor 13f.
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Favorable in vitro potency, pharmacokinetic profiles and in vivo antitumor
efficacy make 13f a promising drug candidate to advance into preclinical
development.